Finding a new pathway for antidepressants

Compiled by BioPhotonics staff

A new fluorescence labeling technique that can monitor the reversible plasma membrane association of protein domains without the need for scanning, optical splicing or imaging has been identified by researchers. The method uses fluoxetine, an active ingredient in Prozac that suppresses the activity of the potassium channel protein TREK1, activity associated with mood regulation.

TREK1 potassium ion channels, fluorescing green in these cultured neurons, have been linked to the regulation of emotions. Images courtesy of Isacoff group, UC Berkeley.

Investigators at the University of California, and at the US Department of Energy’s Lawrence Berkeley National Laboratory found that the previously discovered inhibition of TREK1 by fluoxetine is accompanied by an unbinding of the protein’s C-terminal domain from the membrane. This is the first observation that TREK1 might be regulated by antidepressant drugs.

The TREK1 ion channel, shown in blue, controls the passage of potassium ions (pink) through the plasma membrane (gray) of neurons, which sets neuron excitability.

In the brain, TREK1 acts as a gate to the passage of potassium ions through the neural membrane, which sets the excitability of the neuron. Previous studies conducted in mice showed that, when the TREK1 gene was “knocked out,” the mice displayed a depression-resistant phenotype that mimicked the behavior of mice treated with fluoxetine. It also showed that the antidepressant inhibited the activity of the TREK1 channel. Although the results indicated a possible role of the TREK1 ion channel in response to fluoxetine, the mechanism behind the activity was unclear.

After developing techniques to label the channel proteins with site-specific fluorescent dyes, the investigators detected structural rearrangements of the labeled sites in the channel through changes in the fluorescence. They separated the C-terminal domain from the rest of the protein, tagging it with a GFP (green fluorescent protein). Unlike the TREK1 domain, in which the pore is embedded in the plasma membrane of a neuron, the C-terminal is a short tail that protrudes into the surrounding cytoplasm.

Using voltage clamps to measure electrical currents through the channel and fluorescence to monitor the disposition of the terminal’s domain, the scientists discovered that, when the C-terminal tail was fully bound to the plasma membrane, the TREK1 channel opened more, but when it was unbound from the membrane, the ion channel tended to close. They concluded that fluoxetine causes the isolated C-terminal domain to unbind from the membrane and also causes an inhibition of current from the full TREK1 channel.

The new assay has potential for applications in pharmaceutical research and could be used to follow changes in lipid composition that results from membrane signaling events or to study the binding of membranes by cytoplasmic regulatory domains of ion channels. Future research will be conducted to see how the C-terminal tail is affected by the presence of fluoxetine when it is still attached to the rest of the TREK1 protein.

The research, which appeared in the journal Proceedings of the National Academy of Science (PNAS), Jan. 24, 2011 (doi: 10.1073/pnas. 1015788108), was supported primarily by the National Institutes of Health.